DNMT1 mediates chemosensitivity by reducing methylation of miRNA-20a promoter in glioma cells

Although methyltransferase has been recognized as a major element that governs the epigenetic regulation of the genome during temozolomide (TMZ) chemotherapy in glioblastoma multiforme (GBM) patients, its regulatory effect on glioblastoma chemoresistance has not been well defined. This study investigated whether DNA methyltransferase (DNMT) expression was associated with TMZ sensitivity in glioma cells and elucidated the underlying mechanism. DNMT expression was analyzed by western blotting. miR-20a promoter methylation was evaluated by methylation-specific PCR. Cell viability and apoptosis were assessed using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) and TdT-mediated dUTP-biotin nick end labeling assays, respectively. The results showed that compared with parental U251 cells, DNMT1 expression was downregulated, miR-20a promoter methylation was attenuated and miR-20a levels were elevated in TMZ-resistant U251 cells. Methyltransferase inhibition by 5-aza-2\'-deoxycytidine treatment reduced TMZ sensitivity in U251 cells. In U251/TM cells, DNMT1 expression was negatively correlated with miR-20a expression and positively correlated with TMZ sensitivity and leucine-rich repeats and immunoglobulin-like domains 1 expression; these effects were reversed by changes in miR-20a expression. DNMT1 overexpression induced an increase in U251/TM cell apoptosis that was inhibited by the miR-20a mimic, whereas DNMT1 silencing attenuated U251/TM cell apoptosis in a manner that was abrogated by miR-20a inhibitor treatment. Tumor growth of the U251/TM xenograft was inhibited by pcDNA-DNMT1 pretreatment and boosted by DNMT1-small hairpin RNA pretreatment. In summary, DNMT1 mediated chemosensitivity by reducing methylation of the microRNA-20a promoter in glioma cells.

Arachnoidal fibrosis after subarachnoid hemorrhage / 中华创伤杂志

Objective To study the mechanism of arachnoidal fibrosis after subarachnoid hemor- rhage. Methods Rats were divided into control group, experiment group and treatment group. Radioim- munoassay (RIA) was employed to detect the levels of hyaluronic acid, laminin,type Ⅲ precollagen and type Ⅳ collagen in the arachnoid membrane. In the meantime, arachnoid cell's morphology and collagen distribution in the subarachnoid space were investigated by electron microscope. Results Results of RIA detection showed increase of Type Ⅲ precollagen level (peak at the second week), obvious higher levels of LN and HA but insignificant change of type IV collagen after subarachnoid hemorrhage. However, dexam- ethasone treatment decreased type Ⅲ precollagen level. Electron microscope found that arachnoid cells pres- ented accentruated bioactivity after subarachnoid hemorrhage, with significant increase of arachnoidal colla- gen fibers from one week after suharachnoid hemorrhage, continuing for 3 weeks. Dexamethasone treatment resulted in low density of mitochondria and sparsed arachnoidal collagen fibers. Conclusions Extracellu- lar matrix (ECM) increases in arachnoid membrane after subarachnoid hemorrhage and participates in a- rachnoid fibrosis. Dexamethasoue can relieve arachnoidal fibrosis after subarachnoid hemorrhage, as pro- rides fresh way for prevention and treatment of post hemorrhagic hydrocephalus.